Poly - (l) - glutamic acid drug delivery system for the intravesical therapy of bladder cancer using WGA as targeting moiety.

In the management of bladder cancer, surgical resection of the tumour is usually followed by intravesical instillation of immunomodulatives and/or chemotherapeutics. The purpose of this local intravesical therapy is to eliminate residual malignant cells after surgical intervention. The main limitation of a localised adjuvant therapy is the insufficient concentration of the active pharmaceutical ingredient (API) in malignant cells due to the unique structure of the human urothelium making it an exclusively hard to overcome barrier in the human body. Different strategies such as electromotive drug administration or local hyperthermia are employed to ameliorate intravesical drug uptake. Previous studies on biorecognitive targeting showed promising results for lectin-, especially wheat germ agglutinin (WGA), mediated drug delivery. Here, we present a targeted conjugate that provides enough binding sites for a possible API as well as high cytoadhesive and cytoinvasive potential. The conjugate should comprise the following components: First WGA, as the targeting moiety, second poly-l-glutamic acid (PGA) as a polymeric backbone providing more than 300 possible binding sites for an API and third fluorescein cadaverine (Fc), a fluorescent dye we coupled to PGA rendering the conjugate traceable. After purification via size exclusion chromatography (SEC) the WGA containing and therefore binding conjugate was isolated from the reaction mix. In flow-cytometric and fluorimetric experiments with single cells and cell monolayers, respectively, binding and internalisation of the conjugate representing a high molecular weight (>100kDa) could be demonstrated. Fluorescent PGA without the WGA component showed neither binding nor internalisation potential. Microscopic colocalization studies with cell monolayers and single cells confirmed the cytoadhesive and cytoinvasive potential of the WGA containing conjugate. In accordance with the results of specificity studies the interaction between the conjugate and the cell surface depended solely on the WGA component of the conjugate. With the help of this targeted drug delivery system limiting factors of intravesical adjuvant therapy could eventually be overcome and thereby treatment efficacy of instillative chemotherapy could be improved. In addition, traceable drug conjugates might bear interesting advantages for theranostic purposes in the treatment of bladder cancer.

Overlapping Yet Response-Specific Transcriptome Alterations Characterize the Nature of Tobacco-Pseudomonas syringae Interactions.

In this study transcriptomic alterations of bacterially induced pattern triggered immunity (PTI) were compared with other types of tobacco-Pseudomonas interactions. In addition, using pharmacological agents we blocked some signal transduction pathways (Ca(2+) influx, kinases, phospholipases, proteasomic protein degradation) to find out how they contribute to gene expression during PTI. PTI is the first defense response of plant cells to microbes, elicited by their widely conserved molecular patterns. Tobacco is an important model of Solanaceae to study resistance responses, including defense mechanisms against bacteria. In spite of these facts the transcription regulation of tobacco genes during different types of plant bacterial interactions is not well-described. In this paper we compared the tobacco transcriptomic alterations in microarray experiments induced by (i) PTI inducer Pseudomonas syringae pv. syringae type III secretion mutant (hrcC) at earlier (6 h post inoculation) and later (48 hpi) stages of defense, (ii) wild type P. syringae (6 hpi) that causes effector triggered immunity (ETI) and cell death (HR), and (iii) disease-causing P. syringae pv. tabaci (6 hpi). Among the different treatments the highest overlap was between the PTI and ETI at 6 hpi, however, there were groups of genes with specifically altered activity for either type of defenses. Instead of quantitative effects of the virulent P. tabaci on PTI-related genes it influenced transcription qualitatively and blocked the expression changes of a special set of genes including ones involved in signal transduction and transcription regulation. P. tabaci specifically activated or repressed other groups of genes seemingly not related to either PTI or ETI. Kinase and phospholipase A inhibitors had highest impacts on the PTI response and effects of these signal inhibitors on transcription greatly overlapped. Remarkable interactions of phospholipase C-related pathways with the proteasomal system were also observable. Genes specifically affected by virulent P. tabaci belonged to various previously identified signaling routes, suggesting that compatible pathogens may modulate diverse signaling pathways of PTI to overcome plant defense.

Synergistic targeting/prodrug strategies for intravesical drug delivery--lectin-modified PLGA microparticles enhance cytotoxicity of stearoyl gemcitabine by contact-dependent transfer.

The direct access to the urothelial tissue via intravesical therapy has emerged as a promising means for reducing the high recurrence rate of bladder cancer. However, few advanced delivery concepts have so far been evaluated to overcome critical inherent efficacy limitations imposed by short exposure times, low tissue permeability, and extensive washout. This study reports on a novel strategy to enhance gemcitabine treatment impact on urothelial cells by combining a pharmacologically advantageous prodrug approach with the pharmacokinetic benefits of a glycan-targeted carrier system. The conversion of gemcitabine to its 4-(N)-stearoyl derivative (GEM-C18) allowed for stable, homogeneous incorporation into PLGA microparticles (MP) without compromising intracellular drug activation. Fluorescence-labeled GEM-C18-PLGA-MP were surface-functionalized with wheat germ agglutinin (WGA) or human serum albumin (HSA) to assess in direct comparison the impact of biorecognitive interaction on binding rate and anchoring stability. MP adhesion on urothelial cells of non-malignant origin (SV-HUC-1), and low- (5637) or high-grade (HT-1376) carcinoma was correlated to the resultant antiproliferative and antimetabolic effect in BrdU and XTT assays. More extensive and durable binding of the WGA-GEM-C18-PLGA-MP induced a change in the pharmacological profile and substantially higher cytotoxicity, allowing for maximum response within the temporal restrictions of instillative administration (120 min). Mechanistically, a direct, contact-dependent transfer of stearoyl derivatives from the particle matrix to the urothelial membrane was found to account for this effect. With versatile options for future application, our results highlight the potential offered by the synergistic implementation of targeting/prodrug strategies in delivery systems tailored to the intravesical route.

Lectin bioconjugates trigger urothelial cytoinvasion--a glycotargeted approach for improved intravesical drug delivery.

A unique structural and functional configuration renders the human urothelium, one of the hardest to overcome biological barriers, and accounts for critical shortcomings in the adjuvant localized therapy of bladder cancer and other severe medical conditions. Strategies to improve intravesical drug absorption are urgently sought, but so far have hardly adopted biorecognitive delivery vectors that are more specifically tailored to the natural characteristics of the target site. The efficient cytoinvasion of uropathogenic bacteria, mediated via a mannose-directed FimH lectin adhesin, and malignancy-dependent differences in bladder cell glycosylation point to considerable unrealized potential of lectins as targeting vectors on the molecular/functional and recognitive level. Here, we outline the ability of wheat germ agglutinin (WGA) to induce endocytosis of conjugated payload in human urothelial SV-HUC-1 cells after stable adhesion to internalizing receptors. A panel of model bioconjugates was prepared by covalently coupling one to six WGA units to fluorescein-labeled bovine serum albumin (fBSA). Cytoadhesive capacity was found to directly correlate to the degree of modification up to a critical threshold of on average three targeting ligands per conjugate. The highly specific, glycan-triggered interaction proved essential for endosomal sorting and was followed by rapid (<60min) and extensive (>40%) internalization. fBSA/WGA bioconjugates were processed analogously to the free lectin, irrespective of the significantly higher molecular weight (100-300kD). Durable entrapment of conjugates in acidic, perinuclear compartments without kiss-and-run recycling to the plasma membrane was found in both single cells and monolayers. Our results assign promising potential to glycotargeted delivery concepts in the intravesical setting and offer new perspectives for the application of complex biologicals in the urinary tract.

Process optimization and biocompatibility of cell carriers suitable for automated magnetic manipulation.

There is increasing demand for automated cell reprogramming in the fields of cell biology, biotechnology and the biomedical sciences. Microfluidic-based platforms that provide unattended manipulation of adherent cells promise to be an appropriate basis for cell manipulation. In this study we developed a magnetically driven cell carrier to serve as a vehicle within an in vitro environment. To elucidate the impact of the carrier on cells, biocompatibility was estimated using the human adenocarcinoma cell line Caco-2. Besides evaluation of the quality of the magnetic carriers by field emission scanning electron microscopy, the rate of adherence, proliferation and differentiation of Caco-2 cells grown on the carriers was quantified. Moreover, the morphology of the cells was monitored by immunofluorescent staining. Early generations of the cell carrier suffered from release of cytotoxic nickel from the magnetic cushion. Biocompatibility was achieved by complete encapsulation of the nickel bulk within galvanic gold. The insulation process had to be developed stepwise and was controlled by parallel monitoring of the cell viability. The final carrier generation proved to be a proper support for cell manipulation, allowing proliferation of Caco-2 cells equal to that on glass or polystyrene as a reference for up to 10 days. Functional differentiation was enhanced by more than 30% compared with the reference. A flat, ferromagnetic and fully biocompatible carrier for cell manipulation was developed for application in microfluidic systems. Beyond that, this study offers advice for the development of magnetic cell carriers and the estimation of their biocompatibility.

Characterization of two blood-brain barrier mimicking cell lines: distribution of lectin-binding sites and perspectives for drug delivery.

In the present study plant lectins with distinct sugar specificities were applied to two blood-brain barrier (BBB) mimicking cell lines, namely human ECV304 and porcine brain microvascular endothelial cells PBMEC/C1-2 in order to elucidate their glycosylation pattern and to evaluate the lectin-cell interaction for lectin-mediated targeting. The bioadhesive properties of fluorescein-labeled lectins were investigated with monolayers as well as single cells using fluorimetry and flow cytometry, followed by confirmation of the specificity of binding. For PBMEC/C1-2 layers highest binding capacity was found for wheat germ agglutinin (WGA), followed by Dolichus biflorus agglutinin (DBA) whereas single cell experiments revealed a predominance of DBA only. Analyzing ECV304 monolayers and single cells, WGA yielded the strongest interaction without any changes during cultivation. The binding capacities of the other lectins increased significantly during differentiation. As similar results to primary cells and brain sections were observed, both cell lines seem to be suitable as models for lectin-interaction studies. Thus, an additional focus was set on the mechanisms involved in uptake and intracellular fate of selected lectins. Cytoinvasion studies were performed with WGA for human ECV304 cells and WGA as well as DBA for PBMEC/C1-2 cells. For both lectins, the association rate to the cells was dependent on temperature which indicated cellular uptake.

Real-time measurements of coagulation on bacterial cellulose and conventional vascular graft materials.

The search for a functional, small diameter (<5mm) vascular graft has been ongoing for over 30 years, but yet there is no consistently reliable synthetic graft. The primary mechanisms of graft failure are intimal hyperplasia, poor blood flow and surface thrombogenicity. Bacterial cellulose (BC) became therefore a proposed new biosynthetic vascular graft material. Since conventional methods are not suited for coagulation measurements on BC, we have adapted the automated calibrated thrombin generation method for measurements of biomaterial-induced coagulation of BC as compared with clinically used graft materials i.e., expanded poly(tetrafluoroethylene) (ePTFE) and poly(ethyleneterephtalat) (PET). We have also visualized the coagulation propagation at the material surfaces. Thrombin generation experiments revealed dramatic differences between the materials tested. Both ePTFE and BC were found to generate longer lag times and ttpeak values than PET. Most importantly, BC was found to generate the lowest "peak", indicating a slower coagulation process at the surface. These results are also supported by the measurements of factor XIIa generation and analysis of surface coagulation times, which were detected in the following increasing order (mean + or - SD): PET (27 + or - 8 min)

Alteration of the glycosylation pattern of monocytic THP-1 cells upon differentiation and its impact on lectin-mediated drug delivery.

In the present study, human monocytic THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA) in order to obtain macrophage-like cells. Before and after treatment, plant lectins with distinct sugar specificities were applied in order to elucidate the glycosylation patterns of both monocytic and macrophage-like cell types and to follow changes during differentiation. As a result of flow-cytometric analyses, for untreated as well as for PMA-differentiated cells WGA yielded the highest binding rate without significant changes in the binding capacity. For the other lectins, divergent results were obtained which point to reorganization of sugar residues on the cell surface during differentiation. Additionally, cytoinvasion being beneficial for enhanced drug absorption was studied with WGA which had displayed a high binding capacity together with a high specificity. For both untreated and PMA-differentiated cells decreased fluorescence intensity at 37 degrees C as compared to 4 degrees C was observable pointing to internalization and accumulation within acidic compartments. Moreover, WGA-functionalized PLGA nanoparticles were prepared, and their uptake evaluated. Uptake rates of 55% in case of PMA-differentiated cells suggested that WGA-grafted drug delivery systems might be an interesting approach for treatment of infectious diseases provoked by parasites, facultative intracellular bacteria, or viruses such as HIV.

Strategies to improve drug delivery in bladder cancer therapy.

Bladder cancer is the ninth most common malignancy in the world featuring very high gender variability in occurrence. Current options for bladder cancer therapy include surgery, immunotherapy, chemotherapy and radiotherapy with a trend towards multimodal treatments. However, successful management remains a challenge for urologists and oncologists because of the high risk for recurrence and progression. Particularly in the field of bladder cancer chemotherapy, efficacy of treatment might be improved by advanced drug delivery strategies aimed at prolonged residence time within the bladder cavity and increased permeability of the bladder wall during intravesical instillation. Moreover, a deeper understanding of the biology of bladder carcinogenesis and malignant progression stimulated the development of a new generation of anticancer drugs for targeted therapies that might result in increased treatment specificity together with lower toxic potential and higher therapeutic indices. This review discusses the available strategies for 'targeted therapy', focusing on molecular targets, and for 'controlled delivery', comprising all other approaches towards improved drug delivery.

Alteration of the glycocalyx of two blood-brain barrier mimicking cell lines is inducible by glioma conditioned media.

Recent studies showed that glioma conditioned medium is able to induce blood-brain barrier properties in in vitro models. In this regard, it was investigated whether glioma conditioned medium can also influence the lectin-binding capacity of blood-brain barrier in vitro models. For the presented study cell lines PBMEC/C1-2 and ECV304 were chosen because it was previously shown that glioma conditioned medium was able to induce specific blood-brain barrier properties in these cell lines. Six different plant lectins (WGA, STL, LCA, UEA-I, DBA, PNA) with distinct sugar specificities were applied in order to elucidate the glycosylation patterns of cell line PBMEC/C1-2 and ECV304. Lectin-binding studies were carried out with monolayers as well as with single cells. In the case of PBMEC/C1-2 monolayers, results showed a significant increase of the binding of lectins WGA, STL, UEA-I, DBA and PNA after application of 25 pmol lectin when cultured in media containing soluble factors derived from glioma cell line C6, whereas the binding capacity for LCA remained similar. For ECV304 monolayers, a significant decrease of WGA, STL and LCA was observable, whereas UEA-I binding increased in comparison to cells grown in the corresponding basal growth medium without soluble C6 factors. Single cell studies showed less significant, but similar changes in the lectin-interactions with the cell surfaces. In conclusion, it was shown that soluble factors derived from glioma cell line C6 can modulate the "glycocalyx" of blood-brain barrier mimicking cell lines.

Lectin binding patterns reflect the phenotypic status of in vitro chondrocyte models.

In vitro studies using chondrocyte cell cultures have increased our understanding of cartilage physiology and the altered chondrocytic cell phenotype in joint diseases. Beside the use of primary cells isolated from cartilage specimens of donors, immortalized chondrocyte cell lines such as C-28/I2 and T/C-28a2 have facilitated reproducible and standardized experiments. Although carbohydrate structures appear of significance for cartilage function, the contribution of the chondrocyte glycocalyx to matrix assembly and alterations of the chondrocyte phenotype is poorly understood. Therefore, the present study aimed to evaluate the glycoprofile of primary human chondrocytes as well as of C-28/I2 and T/C-28a2 cells in culture. First, the chondrocytic phenotype of primary and immortalized cells was assessed using real-time reverse transcriptase polymerase chain reaction, immunofluorescence, and glycosaminoglycans staining. Then, a panel of lectins was selected to probe for a range of oligosaccharide sequences determining specific products of the O-glycosylation and N-glycosylation pathways. We found that differences in the molecular phenotype between primary chondrocytes and the immortalized chondrocyte cell models C-28/I2 and T/C-28a2 are reflected in the glycoprofile of the cells. In this regard, the glycocalyx of immortalized chondrocytes was characterized by reduced levels of high-mannose type and sialic acid-capped N-glycans as well as increased fucosylated O-glycosylation products. In summary, the present report emphasizes the glycophenotype as an integral part of the chondrocyte phenotype and points at a significant role of the glycophenotype in chondrocyte differentiation.

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.

OBJECTIVE: To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. METHODS: Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). RESULTS: At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. CONCLUSIONS: Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

Lectin binding studies on C-28/I2 and T/C-28a2 chondrocytes provide a basis for new tissue engineering and drug delivery perspectives in cartilage research.

The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin-cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.

Bridging the gap--biocompatibility of microelectronic materials.

There is an increasing interest in cell-based microelectronic biosensors for high-throughput screening of new products from the biotech pipeline. This requires fundamental knowledge of the biocompatibility of the materials used as the growing support for the cells. Using monolayer-forming Caco-2 cells of human origin, the biocompatibility of silicon wafers coated with various metals, dielectrics and semiconductors was assessed. Besides microscopic inspection, proliferation of cells indicating viability as well as brush border enzyme activity indicating differentiation of adherent growing cells were chosen as parameters to estimate biocompatibility. The type of wafer used for deposition of the coating initially influences the biocompatibility of the final product. Whereas p-doped silicon was fully biocompatible, n-doped silicon reduced the proliferation of cells. Among the different coatings, Al and Ti even increased the cell growth as compared to glass. Culturing the cells for 6 days on coated wafers demonstrated that the differentiation of adhering cells on Ti- and ZrO2-coated wafers was comparable to glass, whereas coatings with Si3N4, Au, Al, and ITO reduced differentiation to 15-35%. In the cases of Au and Si3N4 this effect equilibrated with prolonged culturing. These results demonstrate the importance of a careful selection of the materials used for the production of cell-based biosensors.

Controlled myocardial infarction induced by intracoronary injection of n-butyl cyanoacrylatein dogs: a feasibility study.

Concentrated ethanol has been used to induce controlled myocardial infarct in patients with hypertrophic obstructive cardiomyopathy. We report the acute and early follow-up results of an alternative agent, n-butyl cyanoacrylate (n-BCA) glue, in a dog model. In 11 mongrel dogs, we injected n-BCA into different branches of the left anterior descending artery. Biplane left ventriculogram and coronary angiogram were performed before and after injection. In the surviving animals, we performed programmed stimulation (PS) to test for inducible ventricular tachycardia (VT) 48 days later. Following euthanasia, the removed hearts were studied with computer tomography (CT) and gross and histologic examination. Three dogs were lost before injection. Four dogs died within 2 hr to 4 days, and four animals survived 48 days. Accidental embolization of n-BCA into nontarget vessels was documented in four subjects. In the n-BCA-injected animals, homogeneous circumscribed scar was demonstrated by CT and histology. The glue was confined strictly to the tributary of the injected vessel, infiltrating arterioles of 14 mum. There was intense granulomatous reaction (GR) in the vessel wall and in the surrounding myocardium. Remote areas were unaffected. Monomorphic VT was not inducible with PS. We report a feasibility study of n-BCA injection to selected coronary arteries of dogs to cause controlled myocardial infarction. We demonstrated that the glue does not escape from the target artery through capillaries or small collateral vessels and thus produces a sharply demarcated and homogeneous scar, which is confined strictly to the supply zone of the injected vessel. Improvement of the delivery system is necessary to eliminate inadvertent embolization. Long-term follow-up is needed to study the GR induced by n-BCA.

Phase II study of antineoplaston A10 and AS2-1 in children with recurrent and progressive multicentric glioma : a preliminary report.

OBJECTIVE: To evaluate the response rates, survival and toxicity of treatment with antineoplaston A10 and AS2-1 (ANP) in the first 12 children enrolled in our studies diagnosed with incurable recurrent and progressive multicentric glioma. PATIENTS AND METHODS: The patients' median age was 9 years. Six patients were diagnosed with pilocytic astrocytoma, four with low-grade astrocytoma and one with astrocytoma grade 2. In one case of visual pathway glioma, a biopsy was not performed due to a dangerous location. Patients received ANP intravenously initially and subsequently orally. The average duration of intravenous ANP therapy was 16 months and the average dosage of A10 was 7.95 g/kg/day and of AS2-1 was 0.33 g/kg/day. The average duration of oral ANP was 19 months and the average dosage of A10 and AS2-1 was 0.28 g/kg/day. Responses were assessed by MRI according to the National Cancer Institute's criteria and confirmed by PET scans in some cases. RESULTS: Complete response was accomplished in 33%, partial response in 25%, and stable disease in 33% of patients, and there was no progressive disease. One patient was non-evaluable due to only 4 weeks of ANP and lack of follow-up scans. One patient who had stable disease discontinued ANP against medical advice and died 4.5 years later. Ten patients are alive and well from 2 to >14 years post-diagnosis. Only one case of serious toxicity of reversible tinnitus, of one day's duration, was described. The study continues with accrual of additional patients. CONCLUSION: The results of the present study are favourable in comparison with radiation therapy and chemotherapy. We believe that confirmation of these results through further studies may introduce a new promising treatment for incurable paediatric brain tumours.

Allergen-loaded biodegradable poly(D,L-lactic-co-glycolic) acid nanoparticles down-regulate an ongoing Th2 response in the BALB/c mouse model.

BACKGROUND AND OBJECTIVE: Biocompatible and biodegradable microparticles have gained interest as antigen delivery systems during the recent years. We investigated whether biodegradable poly(d,l-lactic-co-glycolic) acid (PLGA) nanospheres could be used as allergen vehicles for few-shot therapy of type I allergy. METHODS: The major birch pollen allergen Bet v 1 was encapsulated in PLGA nanospheres (PLGA-Bet v 1). We examined the antigenicity and the immune response to PLGA-Bet v 1 in a BALB/c mouse model. RESULTS: The antigenicity of Bet v 1 was largely unaffected by PLGA entrapment. When BALB/c mice were immunized subcutaneously with PLGA-Bet v 1, they formed allergen-specific IgG antibodies, but did not develop hypersensitivity to Bet v 1, as shown by type I skin tests. To evaluate their therapeutic potential, PLGA-Bet v 1 with or without Al(OH)3 or non-entrapped Bet v 1 with Al(OH)3 were used for single-shot treatment of sensitized mice. Both groups treated with PLGA-Bet v 1 developed high levels of Bet v 1-specific IgG2a antibodies (P<0.01), whereas IgG1 levels decreased significantly (P<0.01). Moreover, T cells from mice treated with PLGA-Bet v 1 showed IFN-gamma and IL-10 production. The synthesis of these cytokines was enhanced in the groups where Al(OH)3 had been added to the vaccine formulation. CONCLUSION: Allergen-loaded PLGA nanoparticles modulate an ongoing Th2 response in the BALB/c mouse model, as demonstrated by down-regulation of IgG1 and production of IFN-gamma and IL-10. Our data strongly suggest that PLGA nanospheres can advantageously be used for formulations of allergen extracts or allergen derivatives for the few-shot treatment of type I allergy.

Phase II study of antineoplaston A10 and AS2-1 in patients with recurrent diffuse intrinsic brain stem glioma: a preliminary report.

OBJECTIVE: A phase II study of antineoplaston A10 and AS2-1 was conducted to evaluate the antineoplastic activity in patients with recurrent diffuse intrinsic brain stem glioma. PATIENTS AND METHODS: This report describes the results of treatment of the first 12 patients admitted to the study. Patients received escalating doses of antineoplaston A10 and AS2-1 by intravenous bolus injections. The median duration of treatment was 6 months and the average dosage of antineoplaston A10 was 11.3 g/kg/day and of antineoplaston AS2-1 0.4 g/kg/day. Responses were assessed by gadolinium-enhanced magnetic resonance imaging of the head. RESULTS: Of ten evaluable patients, complete response was determined in two cases (20%), partial response in three (30%), stable disease in three (30%) and progressive disease in two (20%). Survival at 2 years was 33.3%. Currently, of all 12 patients, two (17%) were alive and tumour free for over 5 years since initial diagnosis; one was alive for more than 5 years, and another for more than 4 years from the start of treatment. Only mild and moderate toxicities were observed, which included three cases of skin allergy, two cases of anaemia, fever and hypernatraemia, and single cases of agranulocytosis, hypoglycaemia, numbness, tiredness, myalgia and vomiting. CONCLUSION: The results of this study compared favourably with the responses of patients treated with radiation therapy and chemotherapy. The study continues with accrual of additional patients.